新基因xVAP019、FAM92A1的克隆及功能研究
|
摘要
目的：进行非洲爪蟾新基因xVAP019和其人类同源基因FAM92A1的克隆及功能研究。
方法：以非洲爪蟾(Xenopus laevis)为研究模型，利用3′未端RT-PCR法克隆从非洲爪蟾卵母细胞cDNA表达文库中筛选到的一个高度保守的新序列表达标签(GenBank accession numbers CV973659)的全长基因(xVAP019)，利用生物信息学方法，分析该基因的同源性、保守性、结构域等特点；半定量RT-PCR和爪蟾胚胎整体原位杂交方法用来检测其时空表达；克隆其完整ORF并构建其pGEM T-easy克隆载体，体外转录mRNA和体外合成其Morpholino(反义分子)，显微注射，研究xVAP019基因在体内过表达和下调内源性xVAP019基因表达时对其胚胎发育的影响；克隆xVAPOl9的人类同源基因FAM92A1，RT-PCR方法和DNA序列测定技术鉴定FAM92A1的新的转录变异体，构建其真核表达载体pcDNA3．1-FAM92A1s，以人类癌细胞系为模型，利用MTT分析法、流式细胞仪分析法、Hoechst33258染色法体外分析其过表达时对细胞增殖和凋亡的作用；血清饥饿法使细胞周期同步化，RT-PCR法分析内源性FAM92A1基因和外源性FAM92A1基因在不同细胞周期阶段的表达情况；构建FAM92A1基因融合蛋白表达质粒pFAM92A-EGFP-N1(增强型绿色荧光蛋白)，转染Hela细胞后瞬时表达，结合DAPI染色，在荧光显微镜下观察FAM92A1蛋白的表达及亚细胞定位；RT-PCR法分析检测FAM92A1s过表达时对p53、Rb、p27、caspase3、caspase 8、caspase 9等基因表达的影响，初步分析其影响细胞增殖和凋亡的可能机制；
结果：(1)克隆了一个非洲爪蟾新基因xVAP019，该基因编码一个含有保守结构域DUF1208且功能未知的蛋白质：(2)生物信息学分析发现xVAP019具有高度的保守性，与其人类同源基因FAM92A1等都属于保守结构域DUF1208家族，含有多个CK2和PKC磷酸化位点和一个蜷曲螺旋结构(coiled coil domain)；(3)实验发现xVAP019在爪蟾成体各组织及各发育阶段都有表达：在卵裂期及囊胚期主要表达于动物半球，在原肠期和神经轴胚期表达较广泛，但主要表达于背部外胚层和脊索中胚层及胚孔区，随后可见xVAP019主要表达于背部外胚层和脊索中胚层的衍生结构如头部、脊索等中枢神经系统区域及表皮；(4)xVAP019体内过表达和下调表达时都严重影响爪蟾胚胎发育，有致畸和致死性；(5)鉴定了FAM92A1的2个新的转录变异体FAM92A1-251、FAM92A1-289且构建了FAM92A1及其两个新的转录变异体FAM92A1-251、FAM92A1-289的真核表达载体：(6)检测了PAM92A1s在不同癌细胞系及正常细胞系中的表达，发现FAM92A1-271和FAM92A1-289在癌细胞系及正常细胞系中均表达丰富，而FAM92A1-251表达较低；(7)FAM92A1-271、FAM92A1-251和FAM92A1-289过表达时可影响细胞周期分布，导致S期进入G／M受阻，抑制细胞增殖且可诱导细胞凋亡；(8)RT-PCR检测发现FAM92A1s在S期高表达，在G1和G2／M期表达较低；(9)FAM92A1s过表达时可见p53、Rb、caspase3、caspase 8的表达增加，未见caspase 9的表达；(10)发现FAM92A1蛋白定位于细胞核。
结论：xVAP019基因表达广泛，编码蛋白属于DUF1208保守结构域家族，体内实验表明，xVAP019基因参与脊椎动物胚胎发育过程中外胚层和中胚层的发育。FAM92A1为xVAP019的同源基因，体外实验证明，新基因蛋白FAM92A1属核蛋白，它具有至少9个选择性剪切体，在人卵巢癌细胞株(SKOV3)中过表达的体外实验显示，FAM92A1s过表达可以明显抑制肿瘤细胞生长、改变细胞周期分布、阻止细胞增殖于S期，并可诱导肿瘤细胞凋亡，表明FAM92A1参与细胞增殖和细胞凋亡的调节。其机制可能与肿瘤细胞p53、Rb、p27等基因的表达有关，其诱导凋亡可能通过caspase3和caspase8途径，而与caspase9无关。
Objective: Cloning and characteristics of the Xenopus larevis novel gene xVAP019 and its human homologous gene FAM92A1.
Methods. Xenopus larevis were here used as model animal. The Xenopus larevis novel gene xVAP019 was cloned by 3' end RT-PCR from a 5'express sequence tag (5'EST) (GenBank accession numbers CV973659), which was screened from Xenopus larevis oocyte cDNA pool. Its homologs and conserved domain were gained by bioinformatics analysis. Semi-quantitative RT-PCR and whole-mount in situ hybridization were used to detect the spatial and temporal expression of xVAP019. We cloned the completed read frame (ORF) of xVAP019 and constructed plasmid pGEMT-xVAP019, and which was used to sythesis xVAP019 mRNA and Morpholino in vitro. By microinjection xVAPO19 mRNA and Morpholino in vivo, we studied the impact of xVAP019 overexpression and downregulation on Xenopus larevis embryonic development. Othermore, the human homologue gene FAM92A1 ofxVAP019 was cloned here, and two novel transcript variants of FAM92A1 were found. Express plasmid pcDNA3.1-FAM92Als were constructed and transfected into SKOV3 cells, then cell cycle and apoptosis analysis were done by flow cytometry, MTT analysis, Hoechst 33258 staining. Synchronize cells in different cycle phase, then examined FAM92A1 expression at the mRNA level in different cell cycle phase by semi-quantitative RT-PCR. The construct containing FAM92A1-271 fused with EGFP was introduced into Hela cells by lipofectamine2000 mediated transfect, and add DAPI staining. The results revealed by directed fluorescence microscopy view. The p53, Rb, p27、caspase3、caspase 8、caspase 9 gene expression was analysed by semi-quantitative RT-PCR when FAM92Als over-expression in SKOV3 cells.
Results: (1) We have identified a novel Xenopus gene (xVAP019) encoding a DUF1208 domain containing protein (2) By bioinformatics analysis, we known that xVAP019 belongs to conserved domain DUF1208 family and contained several Protein kinase C (PKC), Casein kinase 2 (CK2) and a coiled coil domain.(3) Using whole-mount in situ hybridization and RT-PCR, we found abundant xVAP019 maternal transcripts at animal hemisphere during the cleavage stages and blastula stages. During gastrulation xVAP019 is differentially expressed with high levels in the animal helf and highest in marginal zone, then is expressed widely at neurula stages with strongest signal in the prospective CNS regions and the epidermal ectoderm. Subsequently xVAP019 was expressed predominantly in the head, the eyes, the otic vesicle, branchial arches, spinal cord, notochord, somites and tailbud. It is absent or very weak in the endoderm. (4)Injected a morpholino oligo complementary to xVAP019 mRNA or injected a caped xVAP019 mRNA caused most of embryos died during gastrulation and neurulation. Overexpression of xVAP019 mRNA also led to eye defect, shorten interocular distance, small body and abnormal pigment formation in parts of the survival embryos. Similar effect was induced by injection of the xVAP019 human homologous gene FAM92Al.(5)We have also identified two novel transcripts variants (FAM92A1-251, FAM92A1-289) of FAM92A1 and constructed their eukaryocyte express plasmid pcDNA3.1 -FAM92A 1-271, pcDNA3.1 -FAM92A 1-251 and pcDNA3.1-FAM92A1-289. (6) We demonstrated the presence of FAM92Als in different cells, and found FAM92Al-271 and FAM92Al-289 were highly expressed in both cancerous and normal cells, while FAM92Al-251 was consistently expressed at a low level. (7) Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis observed as reducing MTT values.(8) Using semi-quantitative RT-PCR, we found that FAM92A1 s express main in S-phase. (9) When FAM92A1 s over-expression, p53,Rb, caspase3,caspase8 have a increased mRNA level,while no caspase9 mRNA was detected. (10)The subcellular location experiment showed that FAM92A1 localizes in nuclear.
Conclusion: The novel gene xVAP019 is high conserved in vertibrates and express in ectoderm and axis mesoderm. Our results suggest that xVAP019 is essential for the normal ectoderm and axis mesoderm differentiation and embryos survival. This investigation is the first in vivo study examining the role of this novel gene and reveals an important role of xVAP019 in embryonic development.
FAM92A1 gene has different transcript variants. FAM92A1 protein is located in nuclear and may take part in the regulation of cell proliferation and apoptosis.
方法：以非洲爪蟾(Xenopus laevis)为研究模型，利用3′未端RT-PCR法克隆从非洲爪蟾卵母细胞cDNA表达文库中筛选到的一个高度保守的新序列表达标签(GenBank accession numbers CV973659)的全长基因(xVAP019)，利用生物信息学方法，分析该基因的同源性、保守性、结构域等特点；半定量RT-PCR和爪蟾胚胎整体原位杂交方法用来检测其时空表达；克隆其完整ORF并构建其pGEM T-easy克隆载体，体外转录mRNA和体外合成其Morpholino(反义分子)，显微注射，研究xVAP019基因在体内过表达和下调内源性xVAP019基因表达时对其胚胎发育的影响；克隆xVAPOl9的人类同源基因FAM92A1，RT-PCR方法和DNA序列测定技术鉴定FAM92A1的新的转录变异体，构建其真核表达载体pcDNA3．1-FAM92A1s，以人类癌细胞系为模型，利用MTT分析法、流式细胞仪分析法、Hoechst33258染色法体外分析其过表达时对细胞增殖和凋亡的作用；血清饥饿法使细胞周期同步化，RT-PCR法分析内源性FAM92A1基因和外源性FAM92A1基因在不同细胞周期阶段的表达情况；构建FAM92A1基因融合蛋白表达质粒pFAM92A-EGFP-N1(增强型绿色荧光蛋白)，转染Hela细胞后瞬时表达，结合DAPI染色，在荧光显微镜下观察FAM92A1蛋白的表达及亚细胞定位；RT-PCR法分析检测FAM92A1s过表达时对p53、Rb、p27、caspase3、caspase 8、caspase 9等基因表达的影响，初步分析其影响细胞增殖和凋亡的可能机制；
结果：(1)克隆了一个非洲爪蟾新基因xVAP019，该基因编码一个含有保守结构域DUF1208且功能未知的蛋白质：(2)生物信息学分析发现xVAP019具有高度的保守性，与其人类同源基因FAM92A1等都属于保守结构域DUF1208家族，含有多个CK2和PKC磷酸化位点和一个蜷曲螺旋结构(coiled coil domain)；(3)实验发现xVAP019在爪蟾成体各组织及各发育阶段都有表达：在卵裂期及囊胚期主要表达于动物半球，在原肠期和神经轴胚期表达较广泛，但主要表达于背部外胚层和脊索中胚层及胚孔区，随后可见xVAP019主要表达于背部外胚层和脊索中胚层的衍生结构如头部、脊索等中枢神经系统区域及表皮；(4)xVAP019体内过表达和下调表达时都严重影响爪蟾胚胎发育，有致畸和致死性；(5)鉴定了FAM92A1的2个新的转录变异体FAM92A1-251、FAM92A1-289且构建了FAM92A1及其两个新的转录变异体FAM92A1-251、FAM92A1-289的真核表达载体：(6)检测了PAM92A1s在不同癌细胞系及正常细胞系中的表达，发现FAM92A1-271和FAM92A1-289在癌细胞系及正常细胞系中均表达丰富，而FAM92A1-251表达较低；(7)FAM92A1-271、FAM92A1-251和FAM92A1-289过表达时可影响细胞周期分布，导致S期进入G／M受阻，抑制细胞增殖且可诱导细胞凋亡；(8)RT-PCR检测发现FAM92A1s在S期高表达，在G1和G2／M期表达较低；(9)FAM92A1s过表达时可见p53、Rb、caspase3、caspase 8的表达增加，未见caspase 9的表达；(10)发现FAM92A1蛋白定位于细胞核。
结论：xVAP019基因表达广泛，编码蛋白属于DUF1208保守结构域家族，体内实验表明，xVAP019基因参与脊椎动物胚胎发育过程中外胚层和中胚层的发育。FAM92A1为xVAP019的同源基因，体外实验证明，新基因蛋白FAM92A1属核蛋白，它具有至少9个选择性剪切体，在人卵巢癌细胞株(SKOV3)中过表达的体外实验显示，FAM92A1s过表达可以明显抑制肿瘤细胞生长、改变细胞周期分布、阻止细胞增殖于S期，并可诱导肿瘤细胞凋亡，表明FAM92A1参与细胞增殖和细胞凋亡的调节。其机制可能与肿瘤细胞p53、Rb、p27等基因的表达有关，其诱导凋亡可能通过caspase3和caspase8途径，而与caspase9无关。
Objective: Cloning and characteristics of the Xenopus larevis novel gene xVAP019 and its human homologous gene FAM92A1.
Methods. Xenopus larevis were here used as model animal. The Xenopus larevis novel gene xVAP019 was cloned by 3' end RT-PCR from a 5'express sequence tag (5'EST) (GenBank accession numbers CV973659), which was screened from Xenopus larevis oocyte cDNA pool. Its homologs and conserved domain were gained by bioinformatics analysis. Semi-quantitative RT-PCR and whole-mount in situ hybridization were used to detect the spatial and temporal expression of xVAP019. We cloned the completed read frame (ORF) of xVAP019 and constructed plasmid pGEMT-xVAP019, and which was used to sythesis xVAP019 mRNA and Morpholino in vitro. By microinjection xVAPO19 mRNA and Morpholino in vivo, we studied the impact of xVAP019 overexpression and downregulation on Xenopus larevis embryonic development. Othermore, the human homologue gene FAM92A1 ofxVAP019 was cloned here, and two novel transcript variants of FAM92A1 were found. Express plasmid pcDNA3.1-FAM92Als were constructed and transfected into SKOV3 cells, then cell cycle and apoptosis analysis were done by flow cytometry, MTT analysis, Hoechst 33258 staining. Synchronize cells in different cycle phase, then examined FAM92A1 expression at the mRNA level in different cell cycle phase by semi-quantitative RT-PCR. The construct containing FAM92A1-271 fused with EGFP was introduced into Hela cells by lipofectamine2000 mediated transfect, and add DAPI staining. The results revealed by directed fluorescence microscopy view. The p53, Rb, p27、caspase3、caspase 8、caspase 9 gene expression was analysed by semi-quantitative RT-PCR when FAM92Als over-expression in SKOV3 cells.
Results: (1) We have identified a novel Xenopus gene (xVAP019) encoding a DUF1208 domain containing protein (2) By bioinformatics analysis, we known that xVAP019 belongs to conserved domain DUF1208 family and contained several Protein kinase C (PKC), Casein kinase 2 (CK2) and a coiled coil domain.(3) Using whole-mount in situ hybridization and RT-PCR, we found abundant xVAP019 maternal transcripts at animal hemisphere during the cleavage stages and blastula stages. During gastrulation xVAP019 is differentially expressed with high levels in the animal helf and highest in marginal zone, then is expressed widely at neurula stages with strongest signal in the prospective CNS regions and the epidermal ectoderm. Subsequently xVAP019 was expressed predominantly in the head, the eyes, the otic vesicle, branchial arches, spinal cord, notochord, somites and tailbud. It is absent or very weak in the endoderm. (4)Injected a morpholino oligo complementary to xVAP019 mRNA or injected a caped xVAP019 mRNA caused most of embryos died during gastrulation and neurulation. Overexpression of xVAP019 mRNA also led to eye defect, shorten interocular distance, small body and abnormal pigment formation in parts of the survival embryos. Similar effect was induced by injection of the xVAP019 human homologous gene FAM92Al.(5)We have also identified two novel transcripts variants (FAM92A1-251, FAM92A1-289) of FAM92A1 and constructed their eukaryocyte express plasmid pcDNA3.1 -FAM92A 1-271, pcDNA3.1 -FAM92A 1-251 and pcDNA3.1-FAM92A1-289. (6) We demonstrated the presence of FAM92Als in different cells, and found FAM92Al-271 and FAM92Al-289 were highly expressed in both cancerous and normal cells, while FAM92Al-251 was consistently expressed at a low level. (7) Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis observed as reducing MTT values.(8) Using semi-quantitative RT-PCR, we found that FAM92A1 s express main in S-phase. (9) When FAM92A1 s over-expression, p53,Rb, caspase3,caspase8 have a increased mRNA level,while no caspase9 mRNA was detected. (10)The subcellular location experiment showed that FAM92A1 localizes in nuclear.
Conclusion: The novel gene xVAP019 is high conserved in vertibrates and express in ectoderm and axis mesoderm. Our results suggest that xVAP019 is essential for the normal ectoderm and axis mesoderm differentiation and embryos survival. This investigation is the first in vivo study examining the role of this novel gene and reveals an important role of xVAP019 in embryonic development.
FAM92A1 gene has different transcript variants. FAM92A1 protein is located in nuclear and may take part in the regulation of cell proliferation and apoptosis.
引文
1. Nieuwkoop and Faber. (1994).Normal table of Xenopus laevis. Garland Publishing, New York.
2. Sive H.L.,R.M.Grainger,and R.M.Harland. (2000). Early development of Xenopus laevis: a laboratory manual by Cold Spring Harbor Laboratory Press. p93-100
3. Summerton J, Weller D. (1997) .Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7(3): 187-95. Review.
4. Heasman J, Kofron M, Wylie C. (2000).Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol. Jun 1;222(1): 124-34.
5. Heasman J. (2002). Morpholino oligos: making sense of antisense? Dev Biol .243(2):209-214. Review
6. Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT. (2004). BAR Domains as Sensors of Membrane Curvature: The Amphiphysin BAR Structure. Science 303:495-499.
7. Yoshida Y, Kinuta M, Abe T, Liang S, Araki K, Cremona O, Di Paolo G, Moriyama Y, Yasuda T, De Camilli P, Takei K. (2004).The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature. EMBOJ., 23(17): 3483 - 3491.
8. David M.,Loppnow,B.,Tiedemann,H.(1987).Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester. Roux's Arch.Dev.Biol.196:137-140.
9. Otte A.P.,Koster,C.H.,Snock,G.T. and Durston,A.J. (1988).Protein kinase C mediates neural induction in Xenopus laevis. Nature 334:618-620.
10. Grunz H.(1999a).Gene expression and pattern formation during early embryonic development in amphibians.J.Biosci.24:515-528.
11. Westmark CJ, Ghose R, Huber PW. (2002). Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2. Biochem J. 362(Pt 2):375-82.
12. Tapia JC, Bolanos-Garcia VM, Sayed M, Allende CC, Allende JE. (2004). Cell cycle regulatory protein p27KIPl is a substrate and interacts with the protein kinase CK2. J Cell Biochem.91(5):865-79.
13. Seldin DC, Landesman-Bollag E, Farago M, Currier N, Lou D, Dominguez I. (2005). CK2 as a positive regulator of Wnt signalling and tumourigenesis. Mol Cell Biochem. 74(1-2):63-7
14. Dominguez I, Mizuno J, Wu H, Song DH, Symes K, Seldin DC. (2004). Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos. Dev Biol. 274(1):110-24.
15. Dominguez I, Mizuno J, Wu H, Imbrie GA, Symes K, Seldin DC. (2005).A role for CK2 alpha/beta in Xenopus early embryonic development. Mol Cell Biochem. 274(1-2): 125-31
1. Marchler-Bauer A, Bryant SH. CD-Search: protein domain annotations on the fly[J]. Nucleic Acids Res., 2004,32: W327-331.
2. Fumihiko Kimura, Shinya Suzu, Yukitsugu Nakamura, et al., Cloning and Characterization of a Novel RING-B-box-Coiled-coil Protein with Apoptotic Function. J. Biol. Chem., Jun 2003; 278: 25046 - 25054.
3. Wang J, Paradis P, Aries A, et al., Convergence of protein kinase C and JAK-STAT signaling on transcription factor GATA-4[J]. Mol Cell Biol. 2005,25(22):9829-44.
4. Kevin C. Corbit, Nicholas Trakul, Eva M. Eves, et al., Activation of Raf-1 Signaling by Protein Kinase C through a Mechanism Involving Raf Kinase Inhibitory Protein. J. Biol. Chem., 2003,278(15): 13061-13068.
5. Gian Luigi Russo, Mariarosaria Tosto, Annalisa Mupo, et al.,Biochemical and Functional Characterization of Protein Kinase CK2 in Ascidian Ciona intestinalis Oocytes at Fertilization: CLONING AND SEQUENCE ANALYSIS OF cDNA FOR αAND βSUBUNITS. J. Biol. Chem., Jul 2004; 279: 33012 - 33023.
6. Mamoru Matsubara, Koiti Titani, Hisaaki Taniguchi, et al., Direct Involvement of Protein Myristoylation in Myristoylated Alanine-rich C Kinase Substrate (MARCKS)-Calmodulin Interaction. J. Biol. Chem., Dec 2003; 278: 48898 - 48902.
7. Matsubara M, Nakatsu T, Kato H,et al, Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca~(2+)/calmodulin.EMBO J. 2004 February 25; 23(4): 712-718.
8． 季少平，药立波．蛋白激酶C与细胞周期．生命科学，2001，01期
9． 刘振杰，刘新光，梁念慈．蛋白激酶CK2在细胞周期调控中的作用．中国药理学通报，2006年11期．
10. S.A. Renshaw, C.E. Dempsey, F.A. Barnes, et al., Three novel Bid proteins generated by alternative splicing of the human Bid gene[J]. J. Biol. Chem. 2004,279: 2846-2855.
11. P.W. Ng, A.G Porter and R.U. Janicke, Molecular cloning and characterization of two novel pro-apoptotic isoforms of caspase,10[J]. J. Biol. Chem. 1999, 274: 10301-10308.
12. M.A. Benedict, Y. Hu, N. Inohara and G. Nunez, Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9[J]. J. Biol. Chem. 2000, 275:8461-8468.
13. Shi, B., Triebe, D., Kajiji, S.et al. Biochem. Biophys. Res. Commun., 1999,254: 779-785.
1. Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogensis[J].Semin Reprod Endocrinol, 1999,17(3):275-290.
2． 马芳，王智彪。肿瘤，一个发育生物学问题。生命科学，2005，17(5)： 434-437．
3. Petruzzelli GJ. The biology of tumor invasion, angiogenesis and lymph node metastasis[J]. Otorhinolaryngol Relat Spec, ORL, 2000,62(4): 178-185.
4． 张春雨。胚胎植入与肿瘤侵润转移的相似性。生命科学，1997，9(3)： 97-99
5. Hanahan D, Weinberg RA. The hallmark of cancer, cell, 2000,100(1):57-70.
6. Wei YQ, Wang QR, Zhao X, et al. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat Med. 2000,6(10): 1160-1166.
7. Wei YQ, Huang MJ, Yang L, et al. Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen. Proc Natl Acad Sci U S A. 2001,98(20): 11545
8. Liu JY, Wei YQ, Yang L, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood. 2003; 102(5): 1815.
9. Lu Y, Wei YQ, Tian L, et al. Immunogene therapy of tumors with vaccine based on Xenogeneic epidermal growth factor receptor. J Immunol. 2003; 170(6): 3162.
10. Su JM, Wei YQ, Tian L, et al. Immunogene Therapy of tumors with vaccine based on chicken homologous matrix mMetalloprotinase-2. Cancer Research. 2003; 63(3): 600.
11． 高进，章静波．癌的侵袭与转移基础与临床．北京：科学出版社，2003， 255-264．
12. Ren J, Dong L, Xu CB, et al.Effect of antisense oligo-dexynucelotide specific to KDR gene on human gastric carcinoma cell.Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ), 2002, 23(4):333-336.
13． 陈铁河，印木泉，朱炳钗等。端粒酶是一种胚胎性肿瘤标志物。基础医学与临床，1999，19(5)：77-79．
14. Wicha M S,Dontu G, A1-Hajj M,et al.Stem cells in normal breast development and breast cancer.Cell Prolif,2003,36(suppl 1):59-72.
15. Singh SK, Charke I D, Terasaki M,et al. Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63(18):5821-5828
16. Singh S K, Charke I D, Hide T, et al.Cancer Stem cells in nervous system tumors. Oncogene,2004,23(43):7267-7273.
17. Nakano I,Himmati H D,Komblum H I. Cancer Stem cells in pekiatric brain tumor.No Shinkei Geka, 2004,32(8):827-834.
18. Growe DL, Parsa B,Sinha U K.Relationships between stem cells and cancer stem cells.Histopathol,2004,19(2):505-509.
19. Rinkenberger JL, Homing S,Klocke B ,et al. Mc121 deficiency results in prei-implantation embryonic lethality [J]. Genes Dev, 2000,14(1):23-27.
20. Quenby S,Brazeau C,Drakeley A,et al.Oncogene and tumour suppressor gene products during trophoblast differentiation in the first trimesterJ.Mol Hum Reprod, 1998,45:477-481.
21. Tetens F, Kliem A, Tscheudschilsuren G,et al. Expressionof proto-oncogenes inbovine prei-implantation blastocysts J. Anat Embryol (Berl),2000,201(5):349-355.
22． 张键，曹宇静，刘为敏，等．基质金属蛋白酶-28在人细胞滋养层细胞与绒毛膜癌细胞系JEG-3细胞中的表达J．科学通报，2002，47(3)：216-220．
23. Alastair Morrison, Linda Ariza-McNaughton, Alex Gould, Mark Featherstone and Robb Krumlauf~*.HOXD4 and regulation of the group 4 paralog genes. Development, 1997, 124, 3135-3146.
24. Morrison, GM and Brickman, J. Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development. Development 2006, 133,2011-2022.
2. Sive H.L.,R.M.Grainger,and R.M.Harland. (2000). Early development of Xenopus laevis: a laboratory manual by Cold Spring Harbor Laboratory Press. p93-100
3. Summerton J, Weller D. (1997) .Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7(3): 187-95. Review.
4. Heasman J, Kofron M, Wylie C. (2000).Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol. Jun 1;222(1): 124-34.
5. Heasman J. (2002). Morpholino oligos: making sense of antisense? Dev Biol .243(2):209-214. Review
6. Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT. (2004). BAR Domains as Sensors of Membrane Curvature: The Amphiphysin BAR Structure. Science 303:495-499.
7. Yoshida Y, Kinuta M, Abe T, Liang S, Araki K, Cremona O, Di Paolo G, Moriyama Y, Yasuda T, De Camilli P, Takei K. (2004).The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature. EMBOJ., 23(17): 3483 - 3491.
8. David M.,Loppnow,B.,Tiedemann,H.(1987).Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester. Roux's Arch.Dev.Biol.196:137-140.
9. Otte A.P.,Koster,C.H.,Snock,G.T. and Durston,A.J. (1988).Protein kinase C mediates neural induction in Xenopus laevis. Nature 334:618-620.
10. Grunz H.(1999a).Gene expression and pattern formation during early embryonic development in amphibians.J.Biosci.24:515-528.
11. Westmark CJ, Ghose R, Huber PW. (2002). Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2. Biochem J. 362(Pt 2):375-82.
12. Tapia JC, Bolanos-Garcia VM, Sayed M, Allende CC, Allende JE. (2004). Cell cycle regulatory protein p27KIPl is a substrate and interacts with the protein kinase CK2. J Cell Biochem.91(5):865-79.
13. Seldin DC, Landesman-Bollag E, Farago M, Currier N, Lou D, Dominguez I. (2005). CK2 as a positive regulator of Wnt signalling and tumourigenesis. Mol Cell Biochem. 74(1-2):63-7
14. Dominguez I, Mizuno J, Wu H, Song DH, Symes K, Seldin DC. (2004). Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos. Dev Biol. 274(1):110-24.
15. Dominguez I, Mizuno J, Wu H, Imbrie GA, Symes K, Seldin DC. (2005).A role for CK2 alpha/beta in Xenopus early embryonic development. Mol Cell Biochem. 274(1-2): 125-31
1. Marchler-Bauer A, Bryant SH. CD-Search: protein domain annotations on the fly[J]. Nucleic Acids Res., 2004,32: W327-331.
2. Fumihiko Kimura, Shinya Suzu, Yukitsugu Nakamura, et al., Cloning and Characterization of a Novel RING-B-box-Coiled-coil Protein with Apoptotic Function. J. Biol. Chem., Jun 2003; 278: 25046 - 25054.
3. Wang J, Paradis P, Aries A, et al., Convergence of protein kinase C and JAK-STAT signaling on transcription factor GATA-4[J]. Mol Cell Biol. 2005,25(22):9829-44.
4. Kevin C. Corbit, Nicholas Trakul, Eva M. Eves, et al., Activation of Raf-1 Signaling by Protein Kinase C through a Mechanism Involving Raf Kinase Inhibitory Protein. J. Biol. Chem., 2003,278(15): 13061-13068.
5. Gian Luigi Russo, Mariarosaria Tosto, Annalisa Mupo, et al.,Biochemical and Functional Characterization of Protein Kinase CK2 in Ascidian Ciona intestinalis Oocytes at Fertilization: CLONING AND SEQUENCE ANALYSIS OF cDNA FOR αAND βSUBUNITS. J. Biol. Chem., Jul 2004; 279: 33012 - 33023.
6. Mamoru Matsubara, Koiti Titani, Hisaaki Taniguchi, et al., Direct Involvement of Protein Myristoylation in Myristoylated Alanine-rich C Kinase Substrate (MARCKS)-Calmodulin Interaction. J. Biol. Chem., Dec 2003; 278: 48898 - 48902.
7. Matsubara M, Nakatsu T, Kato H,et al, Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca~(2+)/calmodulin.EMBO J. 2004 February 25; 23(4): 712-718.
8． 季少平，药立波．蛋白激酶C与细胞周期．生命科学，2001，01期
9． 刘振杰，刘新光，梁念慈．蛋白激酶CK2在细胞周期调控中的作用．中国药理学通报，2006年11期．
10. S.A. Renshaw, C.E. Dempsey, F.A. Barnes, et al., Three novel Bid proteins generated by alternative splicing of the human Bid gene[J]. J. Biol. Chem. 2004,279: 2846-2855.
11. P.W. Ng, A.G Porter and R.U. Janicke, Molecular cloning and characterization of two novel pro-apoptotic isoforms of caspase,10[J]. J. Biol. Chem. 1999, 274: 10301-10308.
12. M.A. Benedict, Y. Hu, N. Inohara and G. Nunez, Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9[J]. J. Biol. Chem. 2000, 275:8461-8468.
13. Shi, B., Triebe, D., Kajiji, S.et al. Biochem. Biophys. Res. Commun., 1999,254: 779-785.
1. Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogensis[J].Semin Reprod Endocrinol, 1999,17(3):275-290.
2． 马芳，王智彪。肿瘤，一个发育生物学问题。生命科学，2005，17(5)： 434-437．
3. Petruzzelli GJ. The biology of tumor invasion, angiogenesis and lymph node metastasis[J]. Otorhinolaryngol Relat Spec, ORL, 2000,62(4): 178-185.
4． 张春雨。胚胎植入与肿瘤侵润转移的相似性。生命科学，1997，9(3)： 97-99
5. Hanahan D, Weinberg RA. The hallmark of cancer, cell, 2000,100(1):57-70.
6. Wei YQ, Wang QR, Zhao X, et al. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat Med. 2000,6(10): 1160-1166.
7. Wei YQ, Huang MJ, Yang L, et al. Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen. Proc Natl Acad Sci U S A. 2001,98(20): 11545
8. Liu JY, Wei YQ, Yang L, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood. 2003; 102(5): 1815.
9. Lu Y, Wei YQ, Tian L, et al. Immunogene therapy of tumors with vaccine based on Xenogeneic epidermal growth factor receptor. J Immunol. 2003; 170(6): 3162.
10. Su JM, Wei YQ, Tian L, et al. Immunogene Therapy of tumors with vaccine based on chicken homologous matrix mMetalloprotinase-2. Cancer Research. 2003; 63(3): 600.
11． 高进，章静波．癌的侵袭与转移基础与临床．北京：科学出版社，2003， 255-264．
12. Ren J, Dong L, Xu CB, et al.Effect of antisense oligo-dexynucelotide specific to KDR gene on human gastric carcinoma cell.Di-si Junyi Daxue Xuebao (J Fourth Mil Med Univ), 2002, 23(4):333-336.
13． 陈铁河，印木泉，朱炳钗等。端粒酶是一种胚胎性肿瘤标志物。基础医学与临床，1999，19(5)：77-79．
14. Wicha M S,Dontu G, A1-Hajj M,et al.Stem cells in normal breast development and breast cancer.Cell Prolif,2003,36(suppl 1):59-72.
15. Singh SK, Charke I D, Terasaki M,et al. Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63(18):5821-5828
16. Singh S K, Charke I D, Hide T, et al.Cancer Stem cells in nervous system tumors. Oncogene,2004,23(43):7267-7273.
17. Nakano I,Himmati H D,Komblum H I. Cancer Stem cells in pekiatric brain tumor.No Shinkei Geka, 2004,32(8):827-834.
18. Growe DL, Parsa B,Sinha U K.Relationships between stem cells and cancer stem cells.Histopathol,2004,19(2):505-509.
19. Rinkenberger JL, Homing S,Klocke B ,et al. Mc121 deficiency results in prei-implantation embryonic lethality [J]. Genes Dev, 2000,14(1):23-27.
20. Quenby S,Brazeau C,Drakeley A,et al.Oncogene and tumour suppressor gene products during trophoblast differentiation in the first trimesterJ.Mol Hum Reprod, 1998,45:477-481.
21. Tetens F, Kliem A, Tscheudschilsuren G,et al. Expressionof proto-oncogenes inbovine prei-implantation blastocysts J. Anat Embryol (Berl),2000,201(5):349-355.
22． 张键，曹宇静，刘为敏，等．基质金属蛋白酶-28在人细胞滋养层细胞与绒毛膜癌细胞系JEG-3细胞中的表达J．科学通报，2002，47(3)：216-220．
23. Alastair Morrison, Linda Ariza-McNaughton, Alex Gould, Mark Featherstone and Robb Krumlauf~*.HOXD4 and regulation of the group 4 paralog genes. Development, 1997, 124, 3135-3146.
24. Morrison, GM and Brickman, J. Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development. Development 2006, 133,2011-2022.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |